Poxvirus containing formulations and process for preparing stable poxvirus containing compositions

ABSTRACT

The present invention relates to a formulation, in particular an aqueous formulation comprising (i) a poxvirus of one of the genera  orthopoxvirus, avipoxvirus, parapoxvirus, capripoxvirus  and  suipoxvirus , (ii) a disaccharide, (iii) a pharmaceutically acceptable polymer and optionally (iv) a buffer. The aqueous formulation is particularly suitable for freeze drying processes resulting in a stable, freeze-dried, poxvirus containing composition. The invention further concerns a method for preparing a freeze-dried, poxvirus containing composition and the thus obtained product.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the US National Phase of PCT/EP02/13434 filed 28Nov. 2002 and claiming the benefit of the priority of the Danish PatentApplication PA2001 01831 filed 10 Dec. 2001.

The present invention relates to a formulation, in particular an aqueousformulation comprising (i) a poxvirus of one of the generaorthopoxvirus, avipoxvirus, parapoxvirus, capripoxvirus and suipoxvirus,(ii) a disaccharide, (iii) a pharmaceutically acceptable polymer andoptionally (iv) a buffer. The aqueous formulation is particularlysuitable for freeze-drying processes resulting in a stable,freeze-dried, poxvirus containing composition. The invention furtherconcerns a method for preparing a freeze-dried, poxvirus containingcomposition and the thus obtained product.

BACKGROUND OF THE INVENTION

The poxviridae comprise a large family of complex DNA viruses thatreplicate in the cytoplasm of vertebrate and invertebrate cells. Inhumans smallpox was by far the most important poxvirus infection. Thecausative agent of smallpox is the variola virus, a member of the genusOrthopoxvirus. Vaccinia virus, also a member of the genus Orthopoxvirusin the family of Poxviridae, was used as live vaccine to immunizeagainst smallpox. Successful worldwide vaccination with Vaccinia virusculminated in the eradication of variola virus (The global eradicationof smallpox. Final report of the global commission for the certificationof smallpox eradication; History of Public Health, No.4, Geneva: WorldHealth Organization, 1980). In the meantime, most of the stocks ofinfectious variola viruses have been destroyed. However, it can not beexcluded that poxviruses inducing smallpox or smallpoxlike diseasesmight again become a major health problem. Thus, it is necessary to bein a position to produce stable vaccines against poxvirus infections, inparticular variola infections, such as vaccines based on vaccinia virus.

In the past vaccinia viruses have also been used to engineer viralvectors for recombinant gene expression and for the potential use asrecombinant live vaccines (Mackett, M., Smith, G. L. and Moss, B. [1982]P.N.A.S. USA 79, 7415–7419; Smith, G. L., Mackett, M. and Moss, B.[1984] Biotechnology and Genetic Engineering Reviews 2, 383–407). Thisentails inter alia DNA sequences (genes), which code for foreignantigens being introduced into the genome of the Vaccinia viruses withthe aid of DNA recombination techniques. If the gene is integrated at asite in the viral DNA which is non-essential for the life cycle of thevirus, it is possible for the newly produced recombinant Vaccinia virusto be infectious, i.e. the virus is able to infect foreign cells andthus to express the integrated DNA sequence (EP 83286 and EP 110385).The recombinant Vaccinia viruses prepared in this way can be used, onthe one hand, as live vaccines for the prophylaxis of infectiousdiseases and on the other hand, for the preparation of heterologousproteins in eukaryotic cells. Other examples for recombinant vacciniaviruses are viruses harboring therapeutic genes such as suicide genes,ribozyme genes or antisense genes.

Modified Vaccinia virus Ankara (MVA) is known to be exceptionally safe.MVA has been generated by long-term serial passages of the Ankara strainof Vaccinia virus (CVA) on chicken embryo fibroblasts (for review seeMayr, A., Hochstein-Mintzel, V. and Stickl, H. [1975] Infection 3, 6–14;Swiss Patent No. 568, 392). Examples for MVA virus strains deposited incompliance with the requirements of the Budapest Treaty are strains MVA572 deposited at the European Collection of Animal Cell Cultures(ECACC), Salisbury (UK) with the deposition number ECACC V94012707, MVA575 deposited under ECACC V00120707 and MVA-BN with the depositionnumber ECACC V00083008.

MVA is distinguished by its great attenuation that is to say bydiminished virulence or infectiosity while maintaining goodimmunogenicity. The MVA virus has been analyzed to determine alterationsin the genome relative to the wild type CVA strain. Six major deletionsof genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31,000 basepairs have been identified (Meyer, H., Sutter, G. and Mayr A. [1991] J.Gen. Virol. 72, 1031–1038). The resulting MVA virus became severely hostcell restricted to avian cells. Furthermore, MVA is characterized by itsextreme attenuation. When tested in a variety of animal models, MVA wasproven to be avirulent even in immunosuppressed animals. Moreimportantly, the excellent properties of the MVA strain have beendemonstrated in extensive clinical trials (Mayr et al., Zbl. Bakt. Hyg.I, Abt. Org. B 167, 375–390 [1987], Stickl et al., Dtsch. med. Wschr.99, 2386–2392 [1974]). During these studies in over 120,000 humans,including high-risk patients, no side effects were associated with theuse of MVA vaccine. Recombinant MVA useful as vaccines have already beenconstructed (see, e.g., WO 97/02355) and are used in clinical trials. WO98/13500 discloses a recombinant MVA containing and capable ofexpressing DNA sequences encoding dengue virus antigens. The foreign DNAsequences were recombined into the viral DNA at the site of a naturallyoccurring deletion in the MVA genome.

An MVA strain showing an even stronger attenuation and enhanced safetycharacteristics is the strain MVA-BN, deposited at the EuropeanCollection of Animal Cell Cultures (ECACC), Salisbury, UK with thedeposition number V00083008.

Besides vaccinia virus other poxviruses have been used as vectors todeliver genetic information into mammalian cells. In this contextreference is made to avipox viruses such as fowlpoxvirus. Fowlpoxvirusescontaining HIV genes in the genome are disclosed in U.S. Pat. Nos.5,736,368 and 6,051,410.

Processes for preparing poxvirus containing compositions suitable asvaccines are known to the person skilled in the art (see for exampleJoklik W. K., Virology (1962),18, 9–18; Richter, K. H., Abhandlungen ausdem Bundesgesundheitsamt (1970), 9, 53–57). The known purificationresults in aqueous, poxvirus containing solutions or in poxviruscontaining sediments. The poxviruses in these solutions and sedimentsare not stable, i.e. the infectivity of the viruses rapidly decreases.However, it is necessary that a vaccine can be stored and distributed ina stabilized form, especially when the vaccines need to be transportedin tropical regions with limited distribution infrastructure. Afreeze-dried product can be stored at temperatures in the range from 4°C. to 25° C. This is a clear advantage compared to the standard storageconditions for liquid formulations, which have to be stored below −20°C. (“Cryopreservation and freeze-drying protocols” Day J, McLellan M;Methods in Molecular Biology, 38, 1995, Humana Press).

Processes for the freeze-drying of poxviruses, in particular vacciniavirus, and virus containing compositions and solutions suitable for thispurpose are known (Burke et al., Critical Reviews in Therapeutic DrugCarrier Systems (1999), 16, 1–83). In general terms freeze-drying of avaccine involves freezing of the vaccine containing aqueous formulationsuitable for freeze-drying, followed by removing water by sublimationunder conditions of reduced pressure and low temperatures and furtherfollowed by removal of water by desorption under conditions of reducedpressure and higher temperatures

The known poxvirus-containing formulations for freeze-drying haveimportant disadvantages. Many of the known vaccinia virus containingcompositions for freeze-drying contain peptone or haemaccel, which areoften of animal origin. However, there are concerns that animal diseasessuch as BSE could be transmitted from animal to man via animal productssuch as peptone, gelatine or haemaccel. Moreover, the poxviruses in theknown virus containing formulations for freeze-drying have not beenpurified. Thus, the poxvirus containing compositions for freeze-dryingknown in the prior art contain inter alia large amounts of proteinsderived from the cells of the cell or tissue culture system and from thebovine serum used during cell cultivation, respectively.

The person skilled in the art also knows freeze-drying compositions thatdo not contain additional compounds of animal origin (which are e.g.peptone or haemaccel). In this case the compositions contain thefollowing compounds alone or in certain combinations: sodium glutamate,sorbitol, lactose, salts, amino acids and glycerin. However, the productobtained after the freeze-drying process is often rather unstable, i.e.the overall loss in virus titer is unacceptably high during storage.Moreover, it has been shown that the poxvirus tends to form aggregatesin some of these formulations and that other compounds precipitatebefore or during freezing.

U.S. Pat. No. 3,577,526 discloses a smallpox vaccine characterized bythe fact that it is made up of a ground virus material of the vaccinedispersed in sucrose. The amount of sucrose is in the range of 20 to40%. The formulation may further comprise 5% dextran. The term groundvirus refers to virus derived from pulps and pustules. Basically, thelymph is ground to break up lumps and separate the liquid from the deadhair and skin. Thus, the protein load of the vaccine preparation is veryhigh and contributes to the stabilization of the virus.

OBJECT OF THE INVENTION

It is an object of the present invention to provide a poxviruscontaining formulation, in particular an aqueous poxvirus containingformulation, for freeze-drying which leads to a stable freeze-driedproduct, wherein the poxvirus is preferably a purified or at leastpartially purified virus. It is a further object of the presentinvention to provide an aqueous poxvirus containing formulation in whichthe poxviruses do not tend to aggregate and in which the components donot precipitate before or during freezing. It is a further object of thepresent invention to provide poxvirus containing formulation, inparticular an aqueous poxvirus containing formulation, comprising lowamounts of non-poxvirus associated proteins. It is a further object ofthe present invention to provide a stable, freeze-dried, poxviruscontaining composition and a method for obtaining said composition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides poxvirus containing formulation, inparticular an aqueous poxvirus containing formulation. The formulation,in particular the aqueous formulation may be suitable for freeze-dryingof said poxvirus. Furthermore, the invention provides the freeze-dried,poxvirus containing product. The formulation according to the presentinvention, in particular the aqueous formulation, comprises thepoxvirus, a disaccharide, a pharmaceutically acceptable polymer andoptionally further a buffer. Although the freeze-dried formulationaccording to the present invention neither contains stabilizingadditives of animal origin such as peptone, gelatine, haemaccel nor highamounts of proteins derived from the system used to amplify the virus(such as cell culture systems), the virus in the formulation issurprisingly stable, i.e. the poxvirus in the freeze-dried compositionremains infectious for long periods of time, even at high storagetemperatures such as room temperature or 37° C.

If not specified otherwise the term “room temperature” as used in thepresent specification corresponds to a temperature of 20 to 25° C.

The poxviruses to be freeze-dried are any poxviruses selected from thegroup consisting of Orthopoxviruses, Parapoxviruses, Avipoxviruses,Capripoxviruses and Suipoxviruses. These viruses might be useful as avaccine for human beings or animals (Virology, 3^(rd) edition, 1995,ed.-in-chief: Fields, B. N.). Particularly preferred poxviruses areviruses of the genera Orthopoxvirus or Avipoxvirus. Preferred examplesof poxviruses belonging to the genus avipoxvirus are canarypoxvirus andfowlpoxvirus. Preferred examples belonging to the family Orthopoxvirusare cowpoxvirus and vaccinia virus.

The poxvirus contained in the formulation according to the presentinvention can be a naturally occurring poxvirus, an attenuated poxvirusor a recombinant poxvirus.

For vaccination of human beings against smallpox the poxvirus in theformulation is preferably a vaccinia virus strain. Examples for vacciniavirus strains suitable for this purpose are the strains Temple ofHeaven, Copenhagen, Paris, Budapest, Dairen, Gam, MRIVP, Per, Tashkent,TBK, Tom, Bern, Patwadangar, BIEM, B-15, Lister, EM-63, New York CityBoard of Health, Elstree, Ikeda and WR. The most preferred vacciniavirus strains are modified vaccinia virus strain Ankara (MVA) and itsderivatives, in particular the strain that has been deposited at ECACCwith the deposition number V00083008 and strain Elstree.

The poxvirus in the formulation according to the present invention ispreferably a poxvirus that is essentially a pathogen in the animal orsubject to be vaccinated. For this purpose it is preferred either to useattenuated virus strains or to use a poxvirus that naturally replicatesin a host species different from the species to be vaccinated and thatis not pathogenic in the heterologous host.

An “attenuated virus” is a virus originating from a pathogenic virus butthat upon infection of the host organism leads to a lower mortalityand/or morbidity compared to the non-attenuated parent virus. Examplesof attenuated poxviruses are known to the person skilled in the art.Most preferred is modified vaccinia virus Ankara (MVA). Typical MVAstrains are MVA 575 and MVA 572 that have been deposited at the EuropeanCollection of Animal Cell Cultures under the deposition numbers ECACCV00120707 and ECACC V 94012707, respectively. Most preferred is MVA-BNor a derivative thereof, which has been described in WO 02/42480(PCT/EP01/13628). The content of this application is included in thepresent application by reference. MVABN has been deposited at theEuropean Collection of Animal Cell Cultures with the deposition numberECACC V00083008.

Examples of poxviruses for which human beings are heterologous hosts andwhich are not pathogenic in human beings are fowlpoxvirus orcanarypoxvirus.

The term “recombinant virus” refers to any virus having inserted intothe viral genome a heterologous gene that is not naturally part of theviral genome. A heterologous gene can be a therapeutic gene, a genecoding for a peptide comprising at least one epitope to induce an immuneresponse, an antisense expression cassette or a ribozyme gene. Methodsto construct recombinant viruses are known to a person skilled in theart. The most preferred poxvirus vector is MVA, in particular MVA 575and MVA-BN (see above)

It is known to the person skilled in the art how poxviruses can beamplified and recovered from infected cell cultures. Generally, in afirst step eukaryotic cells are infected with the poxvirus that isintended to be part of the formulation according to the presentinvention. The eukaryotic cells are cells that are susceptible toinfection with the respective poxvirus and allow replication andproduction of infectious virus. Such cells are known to the personskilled in the art for all poxviruses. For MVA an example for this typeof cells are chicken embryo fibroblasts (CEF) (Drexler I. et al., J.Gen. Virol. (1998), 79, 347–352). CEF cells can be cultivated underconditions known to the person skilled in the art. Preferably the CEFcells are cultivated in serum-free medium. The incubation timepreferably is 48 to 96 hours at 37° C.±2° C. For the infectionpoxviruses are used at a multiplicity of infection (MOI) of 0.05 to 1TCID₅₀ (TCID=tissue culture infectious dose) and the incubation takesplace 48 to 72 hours at the same temperature.

Progress of infection can be observed by looking at cytopathic effects(CPE), typically a significant rounding of the infected cells.

Poxviruses are known to exist in two different forms: poxvirus attachedto cellular membranes in the cytoplasm of the infected cells(intracellular mature virions (IMV)) and viruses that have beenexternalized (extracellular enveloped virions (EEV)) (Vanderplasschen A.et al., J. Gen. Virol. (1998), 79, 877–887). Both viral forms can beused in the formulations according to the present invention. The EEVscan simply be obtained from the supernatant by centrifugation and may bedirectly suspended in an aqueous formulation including a disaccharideand a pharmaceutically acceptable polymer. However, the virus-containingfractions may comprise cellular detritus and other contaminants.Especially for vaccination of human beings it is thus preferred that thevirus is further purified before it is included into a formulationaccording to the present invention. Methods for the purification ofpoxviruses are known to the person skilled in the art. The purificationstep can be e.g. batch centrifugation (e.g. using sucrose cushions) orcontinuous-flow ultracentrifugation (sucrose gradients), ultrafiltration(e.g. cross-flow filtration using a membrane with a pore size biggerthan 500 kDa, but equal or smaller than 0.1 μm), column chromatography(e.g. ion exchange, hydrophobic interaction, size exclusion or acombination) or a combination of some or all of the above (Masuda N. etal., J Bacteriol (1981) 147, 1095–1104).

In order to obtain IMVs the cells have to be harvested in a first stepand disrupted in a second step. If the infected cells are cells that canbe cultivated in suspension culture the infected cells can easily beharvested by centrifugation. If the infected cells are more or lessintact adherent cells it is possible to harvest the cells, i.e. toremove the cells from the culture vial, before subjecting them to thedisruption step. Harvesting methods are known to the person skilled inthe art. Useful techniques are mechanic methods (e.g. by using a rubbercell scraper), physical methods (e.g. freezing below −15° C. and thawingthe culture vessels above +15° C.) or biochemical methods (treatmentwith enzymes, e.g. Trypsin, in order to detach the cells from theculture vessel). If enzymes are used for this purpose the incubationtime should be controlled, since the enzymes might also damage the virusafter prolonged incubation times.

Methods for the disruption of cells are also known to the person skilledin the art. The freezing-thawing method described above already resultsin a partial disruption of the cells. Other known techniques for thedisruption of cells include the use of ultrasound. The ultrasoundtreatment of cells results id a virus containing homogenate.

For vaccination of animals the poxvirus containing homogenate could beused in the formulations according to the present invention. However, itis again preferred to use poxviruses that have been purified at leastpartially. As outlined above such purification methods are known to theperson skilled in the art.

The poxviruses are contained in the formulation, in particular in theaqueous formulation in a concentration range of 10⁴ to 10⁹ TCID₅₀/ml,preferably in a concentration range of e.g. 10⁵ to 5×10⁸ TCID₅₀/ml, mostpreferably in a concentration range of e.g. 10⁶ to 10⁸ TCID₅₀/ml. Theactual concentration depends on the amount of virus to be administeredto the human being or animal, which in turn depends on the type of virusto be administered. For the vaccinia virus strain Elstree a typicalvaccination dose for humans comprises 2.5×10⁵ TCID₅₀. For the vacciniavirus strain MVA-BN a typical vaccination dose for humans comprises1×10⁸ TCID₅₀.

As pointed out above the poxvirus in the formulation according to thepresent invention is preferably a purified or at least partiallypurified virus. The term “purified or at least partially purified virus”refers to the fact that the virus used in the formulation according tothe present invention has a purity that is higher than that of theunpurified virus (“ground virus”) as used in the vaccines used until theeradication of smallpox (such as disclosed in U.S. Pat. No. 3,577,526).Such a higher purity can be obtained e.g by one or more of the followingmethods: batch centrifugation (e.g. using sucrose cushions) orcontinuous-flow ultracentrifugation (sucrose gradients), ultrafiltration(e.g. cross-flow filtration using a membrane with a pore size biggerthan 500 kDa, but equal or smaller than 0.1 μm), column chromatography(e.g. ion exchange, hydrophobic interaction, size exclusion or acombination). Particularly preferred is ultrafiltration and/or batchcentrifugation by using sucrose cushions. In more general terms the term“purified or at least partially purified virus” refers to viruspreparations (such as preparations comprising MVA or Elstree) having atiter of at least 10⁶, preferably of at least 10⁷; more preferably of atleast 10⁸, even more preferably of at least 5×10⁸ TCID₅₀ per mg totalprotein. For strain Elstree typical preparations have a titer of 8×10⁸TCID₅₀ per mg total protein. Methods how to determine the titer of apoxvirus containing preparation are known to the person skilled in theart; one of these methods is outlined in the example section. The totalprotein content is preferably determined according to the method ofKjeldahl (Lynch, J. M. and Barbano, D. M., Kjeldahl nitrogen analysis asa reference method for protein determination in dairy products. J AOACInt. 1999 November–December; 82(6):1389–98. Review). It is to be notedthat the total protein content is the sum of viral proteins and cellularproteins.

It was unexpected that a formulation comprising a purified or partiallypurified virus, a disaccharide and a pharmaceutically active polymer isstable, since it was believed that the large amounts of non-virusprotein in the unpurified virus preparations contributed to thestability of the prior art formulations.

The formulation according to the present invention, in particular theaqueous formulation, comprises a disaccharide. In contrast tomonosaccharides such as glucose which give a good bioprotection duringfreeze-drying but which have a low collapse temperature and oftenfreeze-dry with collapse, disaccharides have been shown to be effectivefreeze-drying protectants displaying higher collapse temperatures thanmonosaccharides.

The disaccharides comprised in the formulations according to the presentinvention are pharmaceutically acceptable disaccharides having acollapse temperature (Tc) in a range of about −25° C. to −35° C. Typicalcollapse temperatures are −31° C. for sucrose, −28.5° C. for trehaloseand −30.5° C. for lactose. Typical collapse temperatures for the entireformulation according to the present invention are preferably in therange of −50° C. to −20° C. Preferred subranges are e.g. −37° C. to −30°C., −36° C. to −31° C. or −35.7 to −31.2° C.

Preferably the disaccharide is selected from the group consisting oftrehalose, lactose and sucrose. Most preferred is sucrose. Thedisaccharide, preferably sucrose, is contained in the formulationaccording to the present invention, in particular the aqueousformulation, preferably in a concentration range of 10–100 g/l, morepreferably in a range of 20–80 g/l, most preferably in a range of 25–60g/l. For sucrose a typical concentration is 45 g/l.

The formulation according to the present invention, in particular theaqueous formulation, further comprises a pharmaceutically acceptablepolymer. The polymer is preferably selected from the group consisting ofdextran and polyvinylpyrrolidon (PVP). The polymer used has to besoluble in the formulation according to the present invention. Ifdextran is used its molecular weight is preferably in the range of20,000 to 100,000, more preferably in the range of 30,000 to 70,000,most preferably in the range of 36,000 to 44,000. The most preferreddextran has an average molecular weight of 40,000. The concentration ofdextran is in the range of 1 to 50 g/l, preferably in a range of 2 to 40g/l or 3 to 30 g/l. Particularly good results have been observed in theranges of 5 to 50 g/l, 5 to 40 g/l or 5 to 30 g/l. Even more preferredis the range of 8 to 30 g/l. The most preferred range is 10 to 27 g/l.An example for a preferred concentration is 18.9 g/l. The preferredconcentrations and concentration ranges of Dextran as shown above, inparticular the range of 5 to 50 g/l and the corresponding subranges,have the particular advantage that the collapse temperature of theformulation is relatively high, which makes it possible to carry out theprocess in an industrial scale. If PVP is used its molecular weight ispreferably in the range of 50,000 to 400,000, more preferably in a rangeof 70,000 to 360,000. The concentration of PVP is in the range of 5 to200 g/l, more preferably in a range of 5 to 100 g/l, most preferably ina range of 10 to 40 g/l.

The formulation according to the present invention, in particular theaqueous formulation, further may comprise a buffer. As pointed out aboveit was one of the objects of the present invention to provide an aqueouspoxvirus containing formulation in which the poxviruses do not aggregateand in which no precipitation occurs upon drying. It has been shownunexpectedly that such undesired effects are correlated with thepresence of a buffer containing phosphate in the aqueous formulation.Examples for phosphate containing buffer are PBS (phosphate bufferedsaline) and phosphate buffer. Consequently, this particular object ofthe invention is solved by an aqueous formulation for freeze-drying thatdoes not contain a phosphate buffer. Thus, the buffer contained in theformulation according to the present invention is preferably selectedfrom the group consisting of TRIS, TBS, MOPS, HEPES and (bi-)carbonatebuffers. The most preferred buffers are TRIS and TBS.

The buffer is used in a concentration that is sufficient to exert therequired buffer capacity. For TRIS buffers the preferred concentrationrange is 1–50 mM; the most preferred concentration is 10 mM. The pH ispreferably adjusted to a value that on the one hand is pharmaceuticallyacceptable for administration in human beings or animals and that on theother hand is not detrimental to the virus. Thus the pH should be in therange of 6.0 to 9.0, more preferably in the range of 7.2 to 7.8. Themost preferred pH is 7.4.

The unexpectedly good results by using a phosphate free buffer areobtained irrespectively of whether the virus in the formulation is anunpurified, purified or partially purified virus. Purified or partiallypurified viruses are preferred.

The formulation according to the present invention, in particular theaqueous formulation may contain salts such as NaCl. Typicalconcentrations for NaCl are in the range of 10 to 200 mM. An example fora preferred NaCl concentration is 140 mM.

The formulation according to the present invention, in particular theaqueous formulation further may comprise L-glutamic acid salts. The saltis preferably a monopotassium salt or a monosodium salt. Theconcentration of the L-glutamic acid salt is preferably in a range of0.05–0.5 g/l, more preferably in a range of 0.1–0.15 g/l.

Some particularly preferred aqueous formulations according to thepresent application are listed in the following table 1. In allformulations listed in table 1 the buffer is 10 mM TRIS, pH 7.4, 140 mMNaCl.

TABLE 1 Modified Vaccinia Virus First Second Third Ankara (MVA) additiveadditive additive Run in [TCID₅₀/ml] [g/l] [g/l] [g/l] table 6 5 × 10⁸25 10.5 0.06 (L- GT 8 (sucrose) (dextran) glutamic acid) 5 × 10⁸ 34.514.5 0.083 (L- GT 8 (sucrose) (dextran) glutamic acid) 5 × 10⁸ 45 18.90.108 (L- GT 9 (sucrose) (dextran) glutamic acid) 5 × 10⁸ 60 25.2 0.144(L- GT 9 (sucrose) (dextran) glutamic acid) 1 × 10⁸ 45 18.9 0.108 (L- —(sucrose) (dextran) glutamic acid) 1 × 10⁸ 45  3.78 0.108 (L- —(sucrose) (dextran) glutamic acid)

The aqueous formulation according to the present invention is suitablefor freeze-drying. For administration the freeze-dried product has to bereconstituted with an appropriate solvent. According to one embodimentsterile water is added to the freeze-dried product in order to bring thecompounds into solution. Preferably, the amount of added watercorresponds more or less to the amount of water that has been eliminatedduring freeze-drying. Thus, according to this embodiment the compositionof the reconstituted product is more or less identical to the initialaqueous formulation. It is therefore within the scope of the presentinvention that the aqueous formulation according to the presentinvention is used as a vaccine. According to an alternative embodimentthe freeze-dried product may also be reconstituted in any otherpharmaceutically acceptable diluent that may be used in appropriateamounts. By way of example the diluent may be water comprising one ormore of the compounds selected from phenol, glycerol and buffer. Theconcentration of phenol in the reconstituted product is e.g. 0.5%. Aspointed out above, the buffer is preferably not a phosphate buffer.

In summary, this aspect of the invention concerns inter alia thefollowing particularly preferred embodiments: (I) A formulation, inparticular an aqueous formulation, comprising or even consisting of apurified or partially purified poxvirus selected from the groupconsisting of Orthopoxviruses, Parapoxviruses, Avipoxviruses,Capripoxviruses and Suipoxviruses, a disaccharide, a pharmaceuticallyacceptable polymer and optionally a buffer, wherein the buffer ispreferably not a phosphate buffer. Preferably the polymer is dextran,preferably in the amounts as specified above. (II) A formulation, inparticular an aqueous formulation, comprising or even consisting of apoxvirus selected from the group consisting of Orthopoxviruses,Parapoxviruses, Avipoxviruses, Capripoxviruses and Suipoxviruses, adisaccharide, a pharmaceutically acceptable polymer and a buffer,wherein the buffer is not a phosphate buffer and wherein the poxvirus ispreferably a purified or partially purified virus. Preferably thepolymer is dextran, preferably in the amounts as specified above. (III)A formulation, in particular an aqueous formulation, comprising or evenconsisting of a poxvirus selected from the group consisting ofOrthopoxviruses, Parapoxviruses, Avipoxviruses, Capripoxviruses andSuipoxviruses, a disaccharide, a pharmaceutically acceptable polymer andoptionally a buffer, wherein polymer is dextran in the amounts asspecified above, by way of example preferably in the range of 5 to 40g/l. Preferably the buffer is not a phosphate buffer. Preferably thepoxvirus is a purified or partially purified virus.

The term “consisting” as used in the context of options (I), (II) and(III) above, refers to formulations consisting of the above-mentionedcompounds only and to formulations containing in addition one or moresalts. Examples of salts that may be added to the formulations (I), (II)and (III) consisting of the above defined compounds are KCl, NaCl,sodium-glutamate. Thus, the term “consisting” in the definition of theabove defined formulations (I), (II) and (III) does not exclude thepossibility to add one or more salts.

By way of example a specific embodiment of the present inventioncomprises an aqueous formulation comprising a purified or partiallypurified poxvirus selected from the group consisting of Orthopoxviruses,Parapoxviruses, Avipoxviruses, Capripoxviruses and Suipoxviruses, adisaccharide, a pharmaceutically acceptable polymer and a buffer,wherein the disaccharide is sucrose in the above specified amounts,wherein the polymer is dextran in the above specified amount and whereinthe buffer is not a phosphate buffer. By way of example another specificembodiment of the present invention comprises an aqueous formulationconsisting of a poxvirus selected from the group consisting ofOrthopoxviruses, Parapoxviruses, Avipoxviruses, Capripoxviruses andSuipoxviruses, a disaccharide, a pharmaceutically acceptable polymer andoptionally a buffer. The poxvirus is preferably a purified or partiallypurified poxvirus. Preferred amounts and examples of the disaccharide,the polymer and the buffer are outlined above.

It is within the skills of the practitioner how such formulations, inparticular an aqueous formulations containing poxviruses can beadministered and which amounts of virus are used for vaccination. Aspointed out above the vaccines might be used to induce an immuneresponse against the poxviruses itself, in particular if attenuated ornon-pathogenic, non-recombinant poxviruses are used. If the poxvirusesare recombinant poxviruses an immune response is additionally raisedagainst the recombinant protein/peptide expressed by the poxvirusvector.

The term “formulation” as used above usually refers to liquidformulations, preferably to aqueous formulations, if not statedotherwise. If the concentrations or concentration ranges are defined in“mM”, “g/l” and so on this is an indication that the respectiveformulation is a liquid or even aqueous formulation. The term “aqueousformulation” relates to those formulations in which the diluent iswater. However, the scope of the present invention also covers those dryformulations that can be obtained from a liquid or even aqueousformulation according to the present invention by removal of the liquid,irrespective of the method that is used for said removal. Thus, theinvention also covers those dry formulations that are obtained bymethods other than freeze-drying.

In particular, the present invention further relates to a method forpreparing a stable, poxvirus containing composition characterized inthat the formulation according to the present invention, in particularthe aqueous formulation is freeze-dried. In the present application theterms “stable, poxvirus-containing composition” and “freeze-driedpoxvirus containing composition” are used interchangeably if not statedotherwise. The term “stable, poxvirus containing composition” is used inthe present application to define poxvirus-containing compositions inwhich the overall loss in virus titer at an incubation temperature of37° C. during 28 days is less than 0.5 logs, preferably less than 0.4logs. A detailed protocol to determine the virus titer and thus theoverall loss in virus titer is given in the example section. However,any other protocol to determine the viral titer can also be used.

Methods of freeze-drying are generally known to the person skilled inthe art (Day, J. and McLellan, M., Methods in Molecular Biology, HumanaPress, (1995) vol. 38).

A freeze-drying process usually consists of the following steps, whichare explained in more detail below:

-   1. Vaccine preparation;-   2. Sample freezing;-   3. Primary drying (sublimation);-   4. Secondary drying (desorption);-   5. Product stoppering and removal;-   6. Vaccine storage;-   7. Reconstitution.

Vaccine preparation: The production and amplification of poxviruses tobe used as vaccines has been explained in detail above. The poxvirusesare optionally purified. The formulation according to the presentinvention is obtained by adding the above defined disaccharides,polymers and, optionally, buffer, L-glutamic acid and optionally furtheradditives to the poxvirus preparation.

Sample Freezing:

Freezing of the sample does immobilize the components in the solution,thereby preventing product foaming when the pressure is reduced.Freezing is a two-step process during which water initially nucleates,followed by growth of ice crystals, resulting in a mixture of ice andsolute concentrate. Ice nucleation is encouraged by reducing thetemperatures and agitating the cooled suspension. In contrast tonucleation, ice growth is encouraged by increasing temperature, therebydecreasing suspension viscosity. Regardless of the precise freezingpattern, proliferation of ice throughout the medium results in anincrease of solute concentration. Biopolymers in solution or suspensionare damaged or inactivated by exposure to these increasingconcentrations of solute. Rapid cooling minimizes exposure of thebioproduct to the concentrate. Above a critical temperature (the glasstransition temperature) the mass viscosity may decrease sufficiently sothat the glass softens and distorts. It dries to form a structurelesssticky residue within the vial. The temperature of the distortion istermed the collapse temperature. More specifically the collapsetemperature is defined as the temperature at which the mobility of thewater in the interstitial region of the matrix becomes significant. Toavoid the distortion the freezing temperature has to be below thecollapse temperature of the aquous formulation. The collapse temperaturecan be determined according to methods known by the person skilled inthe art, e.g. by differential thermal analysis (Jennings, T. A.,“Lyophilization, Introduction and Basic Principles”, Interpharm Press,Denver, Colo., US, 1999, ISBN 1-57491-081-7, pages 132–134).

If the temperature is too low, water diffusion from the virus may beinhibited, and injury by intracellular ice might occur. Consequently,the person skilled in the art will empirically test several freezingtemperatures which are all below the collapse temperature of the aqueousformulation and he will test which temperature leads to a freeze-driedproduct having the highest titer of infectious poxviruses.

Primary Drying (Sublimation):

The primary drying is that part of the freeze-drying process that drivesthe sublimation of the solvent (ice) from the frozen matrix. The primarydrying process starts after the freeze-dryer attains the requiredcondenser temperature and chamber pressure. The pressure in the chamberis usually lower than 1 mbar, preferably lower than 0.2 mbar. Typicallythe pressure is in the range of 0.04 to 0.12 mbar. Throughout thisspecification these conditions are sometimes referred to as “lowpressure”.

The shelf temperature is increased such that sublimation of the ice inthe product matrix occurs and the product temperature is significantlylower than the collapse temperature of the formulation to ensure acompletely frozen matrix throughout the entire primary drying processand to ensure freeze-drying without collapse. The temperature may remainconstant during the whole primary drying process. Alternatively theshelf temperature can be increased continuously during the primarydrying. However, the temperature of the product has to be below thecollapse temperature during the whole primary drying process. At the endof the primary drying the dried product still can contain more than 5%moisture (w/w). In order to obtain a product with a moisture contentthat will not longer support biological growth or chemical reactions itis necessary to carry out a secondary drying step.

Secondary Drying (Desorption):

During secondary drying water vapor is desorbed from the surface of thecake that is formend during primary drying. This is accomplished byincreasing the temperature while the chamber is still at low pressuresso that water is desorbed from the cake surface.

The shelf temperatures for the secondary drying are determined by thestability of the product and may be in the range of 0° C. to +30° C. Theproduct temperature is usually in the range of −5° C. to 30° C. Morepreferred is a temperature in the range of −5° C. to 20° C. Thesecondary drying can be made in two steps. In a first step the producttemperature may be in the range of −5° C. to +15° C., preferably in therange of 0° C. to +10° C., more preferably in the range of 2° C. to +7°C. The second step is made at a higher temperature than the secondarydrying in the first step. The temperature may be in the range from 0° C.to 30° C., preferably in the range of +5° C. to +20° C. Also theresidual moisture content of the formulation depends on the requirementsof the product. Some products need higher, some products lower moisturecontent to achieve best product stability. The optimal residual moisturecontent as well as the time to reach this has to be determinedempirically.

The secondary drying process is continued until the desired moisture isachieved. Methods to determine the moisture of a product are known tothe person skilled in the art. In particular the coulometricKarl-Fischer titration can be used (Jennings, T. A., “Lyophilization,Introduction and Basic Principles”, Interpharm Press, Denver, Colo., US,1999, ISBN 1-57491-081-7, pages 415–418). The residual moisture contentis preferably lower than 5%, more preferably in a range of 0.5 to 4%,even more preferably in a range of 1 to 3%.

Product Stoppering and Removal:

All product-containing vials are sealed according to methods known tothe person skilled in the art. The vials can be sealed under very lowpressure (e.g. 0.04–2.56 mbar) directly in the freeze dryer. It is alsopossible to seal the vials under more or less normal pressure by using achemically inert gas such as nitrogen or helium. Typically the vials maybe sealed in a nitrogen atmosphere at a pressure of 900 mbar. The vialsare closed, preferably by using butyl rubber stoppers. Once the productis stoppered the system can be returned to atmospheric pressure and theshelf can be unloaded. Afterwards, the vials further may be sealed withaluminum caps for long-term storage.

Vaccine Storage:

The freeze-dried product can be stored at room temperature (25° C.) andremains stable for at least 18 weeks, preferably at least 20 weeks morepreferably at least 22 weeks at this temperature. “Stable at a certaintemperature for a certain period of time” means that the loss of viraltiter at this temperature is less than 0.5 logs during this time period.However, if cooling is available it is preferred that the freeze-driedproduct is stored at lower temperatures such as 4° C. Preferably theproduct is stored in the dark. If this is not possible it is preferredto use coloured glass for storage or any other vials, which avoid adetrimental exposure to light.

Reconstitution.

For reconstitution of the freeze-dried product an appropriate amount ofa solvent is added to the freeze-dried product resulting in apharmaceutically acceptable formulation allowing the administration tohuman beings or animals. The solvent is preferably water. Usually thesolvent is added to the formulation in an amount that correspondssubstantially to the amount of solvent lost during the freeze-dryingprocess.

The invention concerns also the freeze-dried product obtained by theprocess according to the present invention.

Thus, the freeze-dried product according to the present inventioncomprises (i) a poxvirus, preferably of the genera orthopoxvirus oravipoxvirus, (ii) a disaccharide, (iii) a pharmaceutically acceptablepolymer and optionally (iv) a buffer, wherein the pox virus, thedisaccharide, the polymer and the buffer are defined as above.

Typical compositions in the freeze-dried product are shown in thefollowing table 2. In all formulations shown in table 2 the amount ofvirus is 5×10⁸ TCID₅₀/ml.

TABLE 2 Amount of DSG [%] in the 20 30  40 aqueous formulation beforefreeze-drying (DSG: 63 g/l dextran MW 36,000–44,000, 150 g/l sucrose,0.36 g/l L- glutamic acid monopotassium salt monohydrate) Sucrose [%] infreeze-dried 54.7 58.7  58.4 product    (36 mg)    (54 mg)    (72 mg)Dextran [%] in freeze-dried 23 24.7  24.6 product  (15.12 mg)  (22.68mg)  (30.24 mg) L-Glutamic acid Mono-  0.13  0.14  0.14 potassium saltMonohydrate (0.0864 mg) (0.1296 mg) (0.1728 mg) [%] in freeze-driedproduct Mean value freeze dried 65.8 92 123.1 cake [mg] (at filling of1.2 ml)

The freeze-dried product according to the present invention is used forthe preparation of a vaccine. To this end the freeze-dried product isresolved/reconstituted as described above and administered to a humanbeing or an animal according to methods known to the person skilled inthe art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the stability of MVA containing freeze-dried formulation ata temperature of 31° C. The tested formulation is GT23 (see examplesection, and table 6). The virus titer in the aqueous formulationaccording to the present invention is determined before freeze-drying.Freeze-drying was made as described for GT23 (see examples and table 6).After freeze-drying the formulation was stored at 31° C. At theindicated time points the freeze-dried formulation was reconstituted andthe viral titer was again determined.

FIGS. 2 and 3 show the results of the same experiment as described inthe legend to FIG. 1 with the exception that the incubation temperaturewas 37° C. in FIG. 2 and 45° C. in FIG. 3.

EXAMPLES

The following examples will further illustrate the present invention. Itwill be well understood by a person skilled in the art that the providedexample(s) in no way may be interpreted in a way that limits theapplicability of the technology provided by the present invention tothis example(s).

Example 1 Freeze-drying of Formulations Containing Modified VacciniaVirus Strain Ankara (MVA)

In this example formulations according to the present inventioncontaining MVA were freeze-dried under different conditions. Thefreeze-dried product was stored at different temperatures. The stabilityof MVA in the preparation was analyzed by determing the titer of MVAafter reconstitution of the freeze-dried product and comparing it withthe MVA titer before freeze-drying. The influence of different storagetimes on the viral titer was determined. The protocol for thedetermination of the titer of a MVA containing formulation is given inexample 2.

1. Experimental Settings:

Vaccine Preparation/Formulations

To test the freeze-drying process according to the present inventionseveral MVA preparations were used. For the freeze-drying experiments(rows GT1-4, 6–10 and 13–15 in table 6) modified vaccinia virus strainAnkara (MVA) was used. The virus was purified by 36%- and 40%-sucrosecushions centrifugation followed by resuspension in 1 mM Tris-buffer atpH 9.

In freeze dryings experiments GT1-4 (table 6) no additives were used.The used buffer system was 10 mM Tris with 140 mM NaCl at pH 7.4. Forthe freeze dryings starting with number GT6 (table 6) differentadditives were used without changing the buffer system.

Two different formulations with different additives were chosen. Theadditives were given to the virus containing solutions by adding adilution buffer. The composition of the dilution buffers used is shownin the following tables 3 and 4. The addition of dilution buffer 1 (DSG)results in an aqueous formulation according to the present invention.The use of dilution buffer 2 (DGG) results in a formulation used forcomparative analysis.

TABLE 3 Concentration in Dilution buffer 1 (DSG) stock solution [g/l]Dextran (MW 36000–44000) 63 Sucrose 150 L-Glutamic acid Monopotassium0.36 salt Monohydrate

TABLE 4 Concentration in Dilution buffer 2 (DGG) stock solution [g/l]Dextran (MW 36000–44000) 63 Glucose 150 L-Glutamic acid Monopotassium0.36 salt Monohydrate

These dilution buffers were used at different concentrations in thefinal formulation. Dilution buffer 1 (DSG) was used at 16.7%, 23%, 30%and 40% (v/v), dilution buffer 2 (DGG) at 20% (v/v). The TCID₅₀/ml ofthe final formulation was adjusted to 5×10⁸ TCID₅₀/ml with aphysiological Tris-buffer (10 mM Tris-buffer; 140 mM NaCl; pH 7.4).

Sample Freezing

The samples were frozen inside the freeze-dryer (Christ freeze dryer,Type Alpha 2–4). For the formulation with sucrose (DSG) the comparisonof different freezing temperatures (−30° C. to −45° C.) showed that thesuspension has to be frozen to −40° C., which is below the collapsetemperature of the formulation, to get a perfect cake structure. Whenfrozen inside the freeze dryer, −40° C. were reached within about 3.5 to4.5 hours (starting at about 20° C.).

Primary Drying

For the formulation with sucrose (DSG) a collapse temperature of about−30 to −37° C. was assumed on account of the collapse temperature ofsucrose (−31° C.). Therefore, the product temperature was adjusted tovalues in the range of −37 to −41° C., to ensure a completely frozenmatrix. Pressures of 0.04 and 0.12 mbar (−50° C. and −40° C. in thephase diagram of water) were used.

The driving force of sublimation during primary drying is the pressuredifference between the product and the condenser of the freeze dryercreated by their temperature differential. A law of nature is that asthe temperature of water is decreased the pressure over that water alsodecreases. A specific temperature of water is always associated with aspecific pressure. The condenser was set to temperatures in the range of−83° C. to −89° C. The pressure in the chamber and the shelf temperatureregulates the product temperature. This indicates that the length of theprimary drying cannot be shortened very easily, because the condensertemperature is fixed. For increasing the product temperature, T_(C) ofthe formulation is the limiting factor.

Secondary Drying

The temperatures for the secondary drying are determined by thestability of the product. Also the residual moisture content of theformulation depends on the requirements of the product. Some needhigher, some lower moisture content to achieve best product stability.The optimal residual moisture content as well as the time to reach thishas to be determined empirically. Since secondary drying starts when theproduct reaches a temperature above 0° C., secondary drying was done intwo steps. In the first step shelf temperature was regulated for somehours (in the range of 4 to 7 hours) in that way that the producttemperature was above 0° C. (in the range of 4 to 6° C.). In that wayall possibly existing ice that remained after primary drying was melted.By using such mild conditions to start the secondary drying damages toproduct will be minimised. Afterwards, the second step was initiated byincreasing the product temperature to values in the range of 18 to 21°C. for 20–30 hours. The time for the second step is strongly dependenton the wanted residual moisture of the freeze-dried product. To obtaindifferent residual moisture contents different times were used. As assayto measure the residual moisture content of the freeze-dried materialthe coulometric Karl-Fischer titration was (Jennings, T. A.,“Lyophilization, Introduction and Basic Principles”, Interpharm Press,Denver, Colo., US, 1999, ISBN 1-57491-081-7, pages 415–418).

Product Stoppering and Removal

All products made during the process development were sealed under verylow pressure (0.04–2.56 mbar) directly in the freeze dryer. The vialswere closed using butyl rubber stoppers. Once the product was stopperedthe system was returned to atmospheric pressure and the shelf wasunloaded. Afterwards, the vials were sealed with aluminum caps forlong-term storage.

Vaccine Storage

An important aspect of the formulation exercise is to produce a vaccinethat is shelf-stable. Factors influencing stability include residualmoisture content, sealing atmosphere composition, and storageconditions, including temperature, humidity, and light.

The different batches produced during the process development were allstored at 4° C. and at room temperature. Furthermore, a few batches werealso stored at 31° C., 37° C. and 45° C.

All samples were stored in the dark.

Reconstitution

The freeze-dried samples were reconstituted with autoclaved Milli-Qwater. More specifically the water (1.2 ml) was added to the sampleusing a syringe. The suspension was mixed by gentle shaking. Thereconstitution takes only a few seconds. The virus titer of thereconstituted product was determined and compared to the virus titerbefore freeze-drying.

Accelerated Stability Test

The stability of the formulation GT23 (see table 6) was assessed at 31°C., 37° C. and 45° C. The viral titer was monitored regularly. Theresults are shown in FIGS. 1 to 3.

2. Results and Conclusion:

MVA was freeze-dried with and without using different additives.

Formulations without additives were shown to be unstable (see table 6).In this context samples were considered as stable when the titer did notdrop more than 0.5 log. Thus, the term “stable at a certain temperaturefor a certain period of time” means that the loss of viral titer at thistemperature is less than 0.5 logs during this time period. A formulation“fails” if the loss of virus titer during the indicated time period atthe indicated temperature is 0.5 logs or more. Formulations comprisingdextran and glucose showed a very low stability at room temperature. Theformulations according to the present invention comprising differentconcentrations of sucrose and dextran proved to be stable. The stabilityof MVA in the formulations according to the present invention is atleast 25 weeks at 4° C. and room temperature.

Detailed information regarding individual experiments is given in table6:

In table 6 it is shown that the stability of the formulations withoutadditives (table 6, GT1-4) was very poor. After only a few weeks theylost 0.5 logs of their original starting titer, which is not acceptable.

The formulation with 20% (v/v) DGG was not acceptable due to a collapseof the material during the freeze-drying process (data not shown). Thiscollapse is explainable by the low T_(C) of glucose, which was notmarkedly increased by the use of dextran, which has a high T_(C) (−11°C.). For primary drying the lowest possible temperature of thefreeze-drying equipment (−45° C.) was used. Therefore, it was notpossible to decrease temperatures below the T_(C) of glucose. Theformulations with 30% (v/v) DGG did not collapse. This phenomenon isprobably due to the higher overall amount of dextran, which increases.T_(C) to a value higher than the temperature used for primary drying.

Although the material did not collapse the stability, especially at roomtemperature, was not satisfying, which might be due to the low solidstate T_(C) of glucose (table 6, GT 10).

Dilution buffer 1 (DSG) was used in most of the experiments. Thestabilization with this additive is very good. Due to the high T_(C)(−31° C.) of sucrose collapse was not a problem with this formulation.The stability of the freeze-dried material was always good (table 6).There were no big differences between the use of 16.7%, 20%, 23%, 28.6%,30% and 40% (v/v) DSG in the formulation. Stability at 4° C. and roomtemperature is proven for all 6 formulations. The freeze-dried productswith 30% and 40% DSG had a slightly better stability.

One of the formulations (process GT 23, see table 6) was analyzed indetail in an accelerated stability test. The results are shown in FIGS.1 to 3 and summarized in the following table 5.

TABLE 5 Loss of titer Loss of 0.5 logs after Temperature [° C.](experimental data) [days] (calculated) 31 0.245 logs in 29 days 59 370.321 logs in 35 days 54 45 0.332 logs in 29 days 44

At 31° C. the vaccine has been stored for about 1 month and still metspecifications (loss in virus titer is less than half a log). But evenat higher temperatures it would be possible to store the vaccine formore than a month, which might be interesting especially for tropicalregions.

For the old smallpox vaccines the WHO recommended a method forestimation of stability. If the vaccine lost less than 1 log within 4weeks at 37° C., it was assumed to be stable for at least one year whenstored at 4° C. (acceptance criterion for use of the old vaccine wasloss of less than 1 log). As shown the formulation GT23 according to thepresent invention fulfills this criterion.

Example 2 Titration of Modified Vaccinia Virus Ankara (MVA)

The titration of Modified Vaccinia virus Ankara (MVA) is performed in aTCID₅₀-based assay using 10-fold dilutions in a 96-well format. At theendpoint of the assay, infected cells are visualised using ananti-vaccinia virus antibody and an appropriate staining solution.

2–3 day old primary CEF (chicken embryo fibroblasts) cells are -dilutedto 1×10⁵ cells/ml in 7% RPMI. 10 fold dilutions are done with 8replicates per dilution. Following dilution, 100 μl are seeded per wellof 96-well plates. Cells are then incubated over night at 37° C. and 5%CO₂.

Dilutions of the virus containing solutions are made in 10-fold steps(10⁻¹ to 10⁻¹² as appropriate) using RPMI without foetal calf serum.Then, 100 μl of every virus sample is added to the cell containingwells. The 96-well-plates are incubated at 37° C. with 5% CO₂ for 5 daysto allow infection and viral replication.

Cells are stained 5 days after infection with a vaccinia virus specificantibody. For the detection of the specific antibody, a horseradishperoxidase (HRP) coupled secondary antibody is used. The MVA specificantibody is an anti-vaccinia virus antibody, rabbit polyclonal, IgGfraction (Quartett, Berlin, Germany #9503-2057). The secondary antibodyis anti-rabbit IgG antibody, HRP coupled goat polyclonal (Promega,Mannheim, Germany, #W4011). The colour reaction is carried out accordingto known techniques.

Every well with cells that are positive in the colour reaction is markedas positive for the calculation of the TCID₅₀.

The titre is calculated by using the formula of Spearman [1] and Kaerber[2]. Because all assay parameters are kept constant, the followingsimplified formula is used:

${{Virus}\mspace{14mu}{{titre}\;\left\lbrack {{TCID}_{50}/{ml}} \right\rbrack}} = 10^{\lbrack{{a + 1},{5 + \frac{x_{a}}{8} + \frac{x_{b}}{8} + \frac{x_{c}}{8}}}\rbrack}$

-   -   a=dilution factor of last column, in which all eight wells are        positive    -   x_(a)=number of positive wells in column a+1    -   x_(b)=number of positive wells in column a+2    -   x_(c)=number of positive wells in column a+3

TABLE 6 Stability data of poxviruses in different freeze-driedformulations Process Additives Freezing Primary drying Secondary dryingStability GT 1 — To −30° C. Pressure: 0.37 mbar Pressure: 0.37 mbarFails after one week at room Shelf temperature: Shelf temperature:temperature and 37° C.  2° C. (for 20 h)  5° C. (for 2.5 h)  8° C. (for1 h) 10° C. (for 2 h) GT 2 — To −40° C. Pressure: 0.12 mbar Pressure:2.56 mbar Fails after 11 days at room product temperature: producttemperature: temperature and 37° C. −13° C. (for 24 h) −4° C. (for 3.5h) 12° C. (for 4 h) GT 3 — To −39° C. Pressure: 0.07 mbar Pressure: 0.07mbar Fails after 23 days at room product temperature: producttemperature: temperature −16° C. (for 24 h) −1° C. (for 2.75 h)  7° C.(for 3.5 h) GT 4 — To −45° C. Pressure: 0.04 mbar Pressure: 0.04 mbarFails after 2 weeks at 4° C. and product temperature: producttemperature: room temperature −37° C. (for 25.5 h) −3° C. (for 6 h) 14°C. (for 26.5 h) GT 6   30% DSG To −44° C. Pressure: 0.04 mbar Pressure:0.04 mbar Formulation is stable product temperature: producttemperature: → at 4° C. for at least 34 weeks −42° C. (for 23 h)  5° C.(for 6 h) → at room temperature for at 16° C. (for 25 h) least 20 weeksGT 7   23% DSG To −44° C. Pressure: 0.04 mbar Pressure: 0.04 mbarFormulation is stable for at product temperature: product temperature:least 46 weeks at 4° C. and −37° C. (for 22.5 h)  0° C. (for 7 h) roomtemperature 13° C. (for 24 h) GT 8 16.7% and To −42° C. Pressure: 0.04mbar Pressure: 0.04 mbar at 4° C. and room temperature   23% DSG producttemperature: product temperature: stable for at least 45 weeks −38° C.(for 24 h)  4° C. (for 8 h) 17° C. (for 21 h) GT 9   30% and To −44° C.Pressure: 0.04 mbar Pressure: 0.04 mbar formulation with 30% DSG:   40%DSG product temperature: product temperature: stable −37° C. (for 25.5h)  4° C. (for 6.5 h) → at 4° C. for at least 57 weeks 17° C. (for 24 h)→ at room temperature for at least 43 weeks formulation with 40% DSG:stable at 4° C. and room temperature for at least 43 weeks GT 10   30%DGG To −35° C. Pressure: 0.04 mbar Pressure: 0.04 mbar Formulation isstable at 4° C. for product temperature: product temperature: 42 weeks−37° C. (for 23 h)  6° C. (for 6.5 h) Formulation fails after 10 weeks19° C. (for 24 h) at room temperature (RT) GT 11   20% DGG To −45° C.Pressure: 0.04 mbar Pressure: 0.04 mbar Formulations failed at 4° C.after product temperature: product temperature: 56 weeks; at RT after 14weeks −41° C. (for 23 h)  5° C. (for 4 h) 19° C. (for 25.5 h) GT 12  30% DSG To −40° C. Pressure: 0.04 mbar Pressure: 0.04 mbar Formulationis stable for at product temperature: product temperature: least 54weeks at 4° C. −37° C. (for 24 h)  4° C. (for 6 h) At 25° C. stable forat least 40 17° C. (for 24 h) weeks GT 13   30% DSG To −40° C. Pressure:0.04 mbar Pressure: 0.04 mbar Formulation is stable for at producttemperature: product temperature: least 36 weeks at 4° C. −38° C. (for19 h)  4° C. (for 15.5 h) −15° C. (for 9 h) 17° C. (for 26.5 h) GT 14  30% DSG To −40° C. Pressure: 0.04 mbar Pressure: 0.04 mbar Formulationis stable for at product temperature: product temperature: least 50weeks at 4° C. −38° C. (for 21.5 h) 15° C. (for 6.5 h) At 25° C. stablefor at least 19 −13° C. (for 17.5 h) 18° C. (for 26.5 h) weeks GT 15  30% DSG To −41° C. Pressure: 0.04 mbar Pressure: 0.04 mbar Formulationfailed after 49 product temperature: product temperature: weeks at 4°C.; −37° C. (for 20 h)  5° C. (for 3 h) At 25° C. stable for at least 1817° C. (for 24.5 h) weeks GT 23   30% DSG To −40° C. Pressure: 0.12 mbarPressure: 0.04 mbar Data see → Accelerated product temperature: producttemperature: stability test in FIG. 1.3 −34° C. (for 21 h)  7° C. (for18 h) 17° C. (for 28 h)

1. A formulation comprising: (a) a purified or partially purifiedpoxvirus selected from the group consisting of Orthopoxviruses,Parapoxviruses, Avipoxviruses, Capripoxviruses, and Suipoxviruses; (b) adisaccharide; (c) a pharmaceutically acceptable polymer; and (d) abuffer excluding a phosphate buffer, wherein the formulation issubstantially free of non-poxvirus associated proteins.
 2. Formulationaccording to claim 1 wherein the buffer excluding a phosphate buffer isselected from the group consisting of TRIS, TBS, MOPS, HEPES, andbicarbonate buffers.
 3. Formulation according to claim 1 wherein thepoxvirus is a vaccinia virus.
 4. Formulation according to claim 3wherein the vaccinia virus is selected from the group consisting ofstrain Elstree and modified vaccinia virus strain Ankara (MVA). 5.Formulation according to claim 1 wherein the poxvirus is a recombinantpoxvirus.
 6. Formulation according to claim 1 wherein the disaccharideis selected from the group consisting of sucrose, lactose and trehalose.7. Formulation according to claim 1 wherein the concentration of thedisaccharide is in a range of 10 to 100 g per liter.
 8. Formulationaccording to claim 1 wherein the pharmaceutically acceptable polymer isselected from the group consisting of dextran and polyvinylpyrrolidone.9. Formulation according to claim 8 wherein the dextran has a molecularweight in the range of 30,000 to 70,000 and has a concentration of 1 to50 g/l.
 10. Formulation according to claim 1 further comprising glutamicacid.
 11. Formulation according to claim 1 wherein the collapsetemperature is in the range of −37° C. to −30° C.
 12. Formulationaccording to claim 1 wherein the poxvirus is an MVA strain or strainElstree, and the disaccharide is sucrose.
 13. Formulation according toclaim 1 wherein the purified or partially purified poxvirus is a virushaving a titer of at least 10⁶ TCID₅₀ per mg total protein.
 14. A stablepoxvirus-containing vaccine which comprises the formulation according toclaim 1 in freeze-dried form.
 15. The stable poxvirus-containing vaccinedefined in claim 14 having a residual moisture content in the range of 1to 3%.
 16. The stable poxvirus-containing vaccine according to claim 14reconstituted with a pharmaceutically acceptable inert solvent.
 17. Amethod of preparing a freeze-dried, stable poxvirus-containing vaccinewhich comprises the following steps: (i) freezing a formulationcomprising: (a) a purified or partially purified poxvirus selected fromthe group consisting of Orthopoxviruses, Parapoxviruses, Avipoxviruses,Capripoxviruses, and Suipoxviruses; (b) a disaccharide; (c) apharmaceutically acceptable polymer; and (d) a buffer excluding aphosphate buffer, to obtain a frozen formulation, wherein theformulation is substantially free of non-poxvirus associated proteins;(ii) primarily drying the frozen formulation obtained according to step(i) under low pressure and at a product temperature allowing sublimationof the ice in the product matrix, wherein the product temperature islower than the collapse temperature of the formulation, to obtain afreeze-dried product and (iii) secondarily drying the freeze-driedproduct obtained according to step (ii) at low pressure and at a producttemperature in the range of 0 to 30° C. until the residual moisture ofthe freeze-dried product is lower than 5%.
 18. The method of preparingthe freeze-dried, stable poxvirus-containing vaccine defined in claim 17wherein according to step (iii) the residual moisture of thefreeze-dried product obtained is in the range of 1 to 3%.
 19. A methodof reconstituting the freeze-dried, stable poxvirus-containing vaccinedefined in claim 17, which comprises the step of dissolving said vaccinein a sufficient amount of a pharmaceutically acceptable inert solvent.20. A method of vaccinating an animal subject, including a human, inneed of said vaccination against a poxviral disease, which comprises thestep of administering to said animal subject, an amount of the stablepoxvirus-containing vaccine defined in claim 16 effective to provide animmune response against said poxviral disease.